Visceral pain is currently the leading cause for patient visits in the U.S and is the main debilitating aspect in irritable bowel syndrome (IBS). The incidence of IBS is higher among individuals who experienced adverse events during childhood, including verbal, physical and sexual abuse, as well as disease of later affected organs. The focus of the parent K01 proposal was a novel mouse model of neonatal colon irritation (NCI) that reproduced the principal characteristic of IBS - colon hypersensitivity in the absence of histopathology. The main hypothesis was that early insult in the viscerosensory system produces a long-lasting alteration in nociceptive processing at the level of the colon, peripheral nerve and DRG. The current proposal aims to broaden the focus of the original K01 award, by adding a model of neonatal stress (neonatal maternal separation [NMS]) to the ongoing studies of NCI in an attempt to gain a more complete understanding of the vulnerabilities of the developing viscerosensory system and the long-lasting deleterious effects that can result from perturbations during the neonatal critical period. The experiments in this application are outlined in two specific aims and are designed to functionally test the hypothesis that NMS produces lifelong visceral hypersensitivity in mice through heightened excitability of peripheral sensory neurons due to changes in expression of specific membrane receptors, which exacerbate the response to experimental colitis. SA1: To test the hypothesis that NMS in mice produces long-lasting colon hypersensitivity and increased excitability/receptor changes in peripheral nerves innervating the distal colon. To test this hypothesis, a) the visceromotor response (VMR) to colorectal distension will be evaluated in adult NMS mice and b) single cell RT-PCR and calcium imaging will be performed on colon DRG neurons to determine changes in expression, function and interactions between TRPV1 and TRPA1, two channels proposed to regulate visceral sensitivity. SA2: To test the hypothesis that NMS enhances susceptibility to experimental colitis and exacerbates inflammation-induced changes in the distal colon and associated peripheral sensory neurons. To test this hypothesis, trinitrobenzene sulfonic acid (TNBS) will be administered to induce colitis and a) the extent of inflammation will be determined by assessing increases in myeloperoxidase (MPO) activity and growth factor production, and b) VMR, single cell RT-PCR and calcium imaging will be repeated to determine how colitis affects the already altered nociceptive processing in the NMS mice. The addition of the NMS model to ongoing studies of NCI allows for comparisons to be made between two different models of neonatal insult with the same behavioral outcome, heightened visceral sensitivity. Discoveries made using these models will allow for the identification of common and/or divergent pathways that underlie visceral hypersensitivity, which could serve as potential therapeutic targets for treatment of IBS and other functional bowel disorders.